Virulent strains of the soil bacterium Agrobacterium tumefaciens are known to infect dicotyledonous plants and to elicit a neoplastic response in these plants. The tumor-inducing agent in the bacterium is a plasmid that functions by transferring some of its DNA into its host plant's cells where it is integrated into the chromosomes of the host plant's cells. This plasmid is called the Ti plasmid, and the virulence of the various strains of A. tumefaciens is determined in part by the vir region of the Ti plasmid which is responsible for mobilization and transfer of the T-DNA. This section is delimited by two 23 base pair direct repeats designated right border and left border, respectively. Any genetic information placed between these two ends may be mobilized and delivered to a susceptible host. The section of DNA of the Ti plasmid which is incorporated into the chromosomes of the host plant is called T-DNA. Once incorporated into a chromosome, the T-DNA genes behave like normal dominant plant genes. They are stably maintained, expressed and sexually transmitted by transformed plants, and they are inherited in normal Mendelian fashion.
The lump of plant tumor tissue that grows in an undifferentiated way at the site of the A. tumefaciens infection is called a crown gall. Cells of crown gall tumors induced by A. tumefaciens synthesize unusual amino acids called opines. Different strains of A. tumefaciens direct the synthesis of different opines by the crown gall cells, and the particular opine induced is a characteristic of the strain which infected the plant. Further, the ability to catabolize the particular opine induced by a given strain is also characteristic of that strain.
Opines are not normally synthesized by A. tumefaciens or by the uninfected host plants. Although it is the T-DNA which codes for the enzymes involved in the synthesis of the opines, the opine synthases, these genes are expressed only in infected plant tissue. This type of expression is consistent with the observation that these genes are under the control of eukaryotic regulatory sequences on the T-DNA.
The most common opines are octopine and nopaline. The opine synthase that catalyzes the synthesis of octopine is lysopine dehydrogenase, and the opine synthase that catalyzes the synthesis of nopaline is nopaline dehydrogenase.
When crown gall cells are put into culture, they grow to form a callus culture even in media devoid of the plant hormones that must be added to induce normal plant cells to grow in culture. A callus culture is a disorganized mass of relatively undifferentiated plant cells. This ability of crown gall cells to grow in hormone-free media is also attributable to the presence of the T-DNA in the transformed host plant cells as also associated with the T-DNA are genes which direct the synthesis of phyto-hormone.
A DNA segment foreign to the A. tumefaciens genetic manipulation will also be transferred to host plant's cells by A. tumefaciens. Thus, the Ti plasmid can be used as a vector for the genetic engineering of host plants. Although, in wild type A. tumefaciens there is only one Ti plasmid per bacterium, in genetically-engineered A. tumefaciens, the vir region and the T-DNA do not have to be carried on the same Ti plasmid for transfer of the T-DNA to occur. The vir region and the T-DNA can be carried on separate plasmids contained within the same Agrobacterium.
Since monocotyledonous plants do not form crown galls, it has been assumed that the host range of A. tumefaciens was limited to the dicotyledons, and that transformation of monocotyledons could not be effected with this bacterium. Indeed, to date, no one has reported the transformation of any member of the monocotyledonbus Gramineae family, including corn (Zea mays), by infection with A. tumefaciens.
However, recently, Hooykaas-Van Slogteren et al., in Nature, 311, 763 (1984), reported the production of small swellings at wound sites infected with A. tumefaciens on monocotyledons of the Liliaceae and Amaryllidaceae families. Opines were detected in plant cells taken from the wound sites of the infected plants.
Also, Hernalsteens et al. reported in The EMBO Journal, 3, 3039 (1984) that cultured stem fragments of the monocotyledon Asparagus officinalis, a member of the family Liliaceae, infected with A. tumefaciens strain C58 developed tumorous proliferations. One of these tumorous proliferations could be propagated on hormone-free medium, and opines were detected in the established callus culture derived from this tumorous proliferation.
Finally, Lorz et al. in Mol. Gen. Genet., 199, 178 (1985) and Portrykus et al. in Mol. Gen. Genet, 199, 183 (1985) have reported the transformation of Gramineae by direct gene transfer to protoplasts. Protoplasts are plant cells from which the cell wall has been removed by digestion with enzymes. Lorz et al. transformed protoplasts of Triticum monococcum using a DNA construct containing the nopaline synthase promotor and the polyadenylation regulatory signal of the octopine synthase gene.
To obtain transformed plants from the transformed cells generated using either the infection techniques of Hooykaas-Van Slogteren et al. and Hernalsteens et al. or the direct gene transfer techniques of Lorz et al. and Portrykus et al., the plants would have to be regenerated from protoplasts or single cell cultures. However, no one has yet been able to regenerate plants from protoplasts or single cell cultures of the Gramineae, including corn. Indeed, no means of producing transformed corn plants or other transformed differentiated corn organs or tissues is currently known, and no method yet exists for transforming corn in a manner which allows for the expression of exogenous DNA in agriculturally important forms or parts of corn such as seeds, pollen, ears or plants.